We have observed the tumor suppressor protein p53 to be present in cytoplasm and nucleus in a family of at least eleven isoforms that differ in charge but not molecular weight suggesting these isoforms are the product of post-translational modification. We have also shown by two dimensional gel electrophoretic analysis that the isoforms are increasingly acidic, presumably by addition of phosphorus, suggesting an array of precursor molecules at various stages of completion for rapid utilization when signaled for transduction to the nucleus. In addition,we have found in human mammary tumor cells at least 5 HSP70 and 3 species of a highly basic 90Kda protein which immunoprecipitates with a monoclonal antibody for HSP90 are complexed with p53 in the cytoplasm and are co-transported to the nucleus. Once inside the nucleus these proteins are released indicating a conformational change occurring in either or both families of proteins. We plan to isolate the p53 complex from murine and human modified cellular systems. We have developed a baculovirus system for the production of large quantities of wild-type human p53 protein, as well as an immunoaffinity column using the hybridoma P122 for purification of p53. Analysis by two-dimensional gel electrophoresis and fluorography will separate which proteins are associated with p53 in addition to the known HSP species which will act as biomarkers. We plan to utilize human cell lines constructed from the N-terminal and C-terminal truncated human p53 genes as well mouse constructs with mutated p53 nuclear localization signals to allow for cytoplasmic accumulation of complexed proteins due to inhibition of apoptosis since the complex will not translocate to the nucleus.